The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa3 and c. Using a sib-selection procedure we have isolated the cyt-12+ allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12+ allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa3.

Summary

The ADP/ATP translocator is an abundant protein of the mitochondrial inner membrane, which in fungi and mammals is synthesized without a presequence. Here we report that the translocator from potato has an amino-terminal extension which may function in mitochondrial targeting. Several cDNA clones encoding the nucleotide sequence of the ADP/ATP translocator have been isolated from potato leaf and tuber cDNA libraries constructed in lambda phages. Only one class of cDNA clones was found but possibly different translocator genes are expressed in other tissues. High levels of transcripts for the translocator are found in all tissues analysed. Sequence determination of the complete insert of one of the clones reveals a long open reading frame of 1158 bp encoding a protein of 386 amino acids corresponding to a calculated molecular weight of 42 kDa. In contrast, the ADP/ATP translocator proteins from fungi and mammals are significantly smaller. Comparison of the Neurospora translocator with the potato protein shows about 75% sequence homology, being confined to the region after amino acid 85 of the potato polypeptide. Antibodies directed against the fungal translocator recognize a protein of 30 kDa in the inner membrane of potato mitochondria, suggesting that the mature protein has a similar size as the translocators from fungi and mammals. Thus, the additional segment of the potato ADP/ATP translocator forms an amino-terminal extension which may be involved in the import of the protein into plant mitochondria.

A central problem in our understanding of mitochondrial (mt) function remains the question of how coordinate transcriptional control is accomplished between nucleus and mitochondria. Here, we report the initial characterization of a protein of previously unknown function, the product of the YMR030 W gene, that appears to mediate such coordinate gene expression. Expression of YMR030 W is glucose-repressible; a deletion mutant for this gene shows a severe growth defect on glycerol-, but not glucose- or ethanol-based medium. In that mutant, transcript levels from GUT1 and GUT2 are highly attenuated compared with those of the wild-type parent when both are grown on glycerol-based medium. Under the same growth conditions, transcripts from the mt OLI1 gene, which has one copy of a mt upstream activating sequence (UAS) in its 5′-flanking region, are attenuated in the ΔYMR030 W mutant, but mRNA from the mt COX3 (OXI2) gene, which lacks the mt UAS, are not. Some nuclear genes encoding mt-related proteins also show low transcript levels in the ΔYMR030 W mutant in comparison with those of the wild-type parent strain during glycerol-based growth. Localization of the protein, via its expression fused to green fluorescent protein, indicates that it is present in both nucleus and mitochondria, supporting a respiration-related transcriptional role for this gene product in both cellular genetic compartments. Because of its role in both respiratory growth and mt function, we designate the YMR030 W coding sequence RSF1 (respiration factor 1).

An increase in the unspliced cox2 transcript and accompanying decrease in the frequency of RNA editing near the exon/intron junction (intron binding site 1, IBS1) have been reported in cold-treated wheat. Here, an attempt was made to clarify whether a similar phenomenon occurs in rice. Levels of unspliced cox2 transcript increased and its editing at the IBS was abolished after cold treatment. The accumulation of COXII protein remained unaffected. The accumulation of intron-containing transcripts of another eight mitochondrial genes, 23 introns in total, was analyzed by Northern blotting and semi-quantitative RT-PCR. An increase in 14 of the 23 intron-adjoining cDNA after cold treatment was observed. Six RNA editing sites in the IBS of four genes were tested as to their status by sequencing cDNA. One of these sites in the nad7 transcript showed a close association with splicing, with editing and splicing occurring simultaneously, irrespective of cold treatment. Two other sites in the intron-containing cox2 and rps3 transcripts were sensitive to cold, where editing frequency began to decrease 1 day after cold treatment, and finally exhibited a tight association with splicing 14 days later. The other sites were efficiently edited. The intron-spliced transcripts were fully edited at all six sites.

The mitochondrial genome of mitochondrial type (mitotype) SW3 of carrot (Daucus carota L.) encodes intact reading frames for a RNA polymerase (Rpo) and a DNA polymerase (Dpo) similar to those encoded by linear mitochondrial plasmids from plants. A BLAST search of translated nucleotide sequences in GenBank revealed previously unreported plasmid-like Rpo or Dpo sequence fragments in many plant mitochondrial DNAs. Phylogenetic analyses of the relationships between mitochondrial (mt)DNA-encoded and plasmid-encoded Rpos and Dpos from plants suggest that the mitochondrial sequences were derived from integrated plant plasmid sequences. A linear mitochondrial plasmid was detected in a different mitotype (FG21) of carrot by Southern hybridization of the Rpo and Dpo to undigested mtDNAs. Transcripts of the mtDNA-encoded Rpo and Dpo in mitotype SW3 were detected by RT-PCR.

Abstract.

TIM9 has been identified as an additional novel gene required for the petite-positive phenotype in Saccharomyces cerevisiae. tim9-1 was obtained through a screen for respiratory-deficient strains that are unable to survive in the absence of mitochondrial DNA. A point mutation found in the tim9-1 coding region converts codon 71 from Gly to Arg. Examination of genes encoding other Tim components indicated that the temperature-conditional alleles of essential genes for the viability of S. cerevisiae, TIM9, TIM10 and TIM12, are required for petite survival, while deletion of TIM8 and TIM13 has no notable effect on petite cell viability. Northern hybridization results suggested that the Spt7 transcription factor is strictly involved in transcription of TIM9 and that the synergistic lethality of tim9-1/spt7Δ dual mutations is due to the deficiency of TIM9 transcription together with defective function of the tim9-1 protein.

Mitochondrial RNA polymerases (mtRNAPs) are necessary for the biogenesis of mitochondria and for proper mitochondrial function since they transcribe genes on mtDNA for tRNAs, rRNAs, and mRNAs. The unique type of RNA editing identified in mitochondria of Physarum polycephalum is thought to be closely associated with transcription, and as such, RNA editing activity would be expected to be closely associated with the mtRNAP. In order to better characterize the role of mtRNAPs in mitochondrial biogenesis and to determine the role of the Physarum mtRNAP in RNA editing, the cDNA of the Physarum mtRNAP was identified using PCR and degenerate primers designed from conserved motifs in mtRNAPs. This amplification product was used to screen a cDNA library for the cDNA corresponding to the Physarum mtRNAP. A cDNA corresponding to a 3.2 kb transcript containing a 997 codon open reading frame was identified. The amino acid sequence inferred from the open reading frame contains motifs characteristic of mtRNAPs. To confirm that a cDNA for an RNA polymerase had been isolated, the cDNA was expressed in E. coli as an N-terminal maltose binding protein (MBP) fusion protein. The fusion protein was purified by affinity chromatography and shown to have DNA-directed RNA polymerase activity. This functional mtRNAP will be useful for in vitro studies of mitochondrial transcription and RNA editing.

Three respiratory-deficient mutants of cytochrome oxidase subunit I in the yeast mitochondrion have been sequenced. They are located in, or near, transmembrane segment VI, the catalytic core of the enzyme. Respiratory-competent revertants have been selected and studied. The mutant V244M was found to revert at the same site in valine (wild-type), isoleucine or threonine. The revertants of the mutant G251R were of three types: glycine (wild-type), serine and threonine at position 251. A search for second-site mutations was carried out but none were found. Among 60 revertants tested, the mutant K265M was found to revert only to the wild-type allele.

Summary

The nucleotide sequence of kalilo, a linear plasmid that induces senescence in Neurospora by intergrating into the mitochondrial chromosome, reveals structural and genetic features germane to the unique properties of this element. Prominent features include: (1) very long perfect terminal inverted repeats of nucleotide sequences which are devoid of obvious genetic functions, but are unusually GC-rich near both ends of the linear DNA; (2) small imperfect palindromes that are situated at the termini of the plasmid and are cognate with the active sites for plasmid integration into mtDNA; (3) two large, non-overlapping open-reading frames, ORF-1 and ORF-2, which are located on opposite strands of the plasmid and potentially encode RNA and DNA polymerases, respectively, and (4) a set of imperfect palindromes that coincide with similar structures that have been detected at more or less identical locations in the nucleotide sequences of other linear mitochondrial plasmids. The nucleotide sequence does not reveal a distinct gene that codes for the protein that is attached to the ends of the plasmid. However, a 335-amino acid, cryptic, N-terminal domain of the putative DNA polymersse might function as the terminal protein. Although the plasmid has been co-purifed with nuclei and mitochondria, its nucleotide composition and codon usage indicate that it is a mitochondrial genetic element.

Summary

The maternally inherited [exn-5] mutant of Neurospora crassa is characterized by its slow-growth rate and deficiency of cytochrome aa3 relative to wildtype strains. We have determined the DNA sequence of the COXI and COXII genes of the mutant, which encode subunits 1 and 2 of cytochrome c oxidase, respectively. No changes in the DNA sequence of the COXI gene relative to the corresponding wild-type gene were found. In the region of the COXII gene we found two alterations, one a C to T transition eight base pairs upstream of the coding sequence and the second within the coding sequence for subunit 2 affecting amino acid 27 of the precursor polypeptide (amino acid 15 of the mature polypeptide). The altered codon in [exn-5] specifies an isoleucine residue rather than the wild-type threonine residue. The corresponding position in subunit 2 sequences of all other organisms examined is conserved either as a threonine or a serine residue. Thus, we consider it likely that the mutation directly affecting the coding sequence of the polypeptide is responsible for the [exn-5] phenotype. Analysis of serially passaged heterokaryons constructed between wild-type and [exn-5] shows that both mutations segregate with the [exn-5] phenotype. Examination of mitochondrial translation products in [exn-5] revealed a deficiency of subunit 2, as well as the presence of a polypeptide that corresponds to a previously described precursor of subunit 1 that accumulates in a COXI mutant of N. crassa, [mi-3]. We propose possible relationships between [exn-5], [mi-3], and the nuclear su-1[mi-3] allele, which suppresses both mutations.